Oxidative processes, which are in many cases enhanced when chemical modifications are introduced that lower the oxygen affinity, can limit the safety of the HBOC proteins. We have carried out a systematic evaluation of several HBOCs both from human and bovine sources. We have measured both the oxidative products present in the Hb preparations and followed the oxidative reactions during 37oC incubations. Autoxidation, the primary oxidative reaction which initiates the oxidative cascade is highly correlated with P50 (R = 0.987; p< 0.002). However, when comparing the results for the other oxidative processes, two different classes of oxidative reactions are identified. The formation of oxyferrylHb like the rate of autoxidation increases for all modified Hbs. However, the subsequent reactions, which lead to heme damage and eventually heme degradation are enhanced for the modified human Hbs, but actually suppressed for bovine modified Hbs. The rhombic heme (distorted geometry) measured by electron paramagnetic resonance, which is the initial step that causes irreversible damage to the heme, is found to be a reliable measure of the stability of ferrylHb and the tendency to produce degradation products. DBBF-Hb, a Hb-based oxygen carrier (HBOC), for which toxic side effects have been well documented, has the highest level of rhombic heme (41-fold greater than for HbAo), even though its rate of autoxidation is relatively low. These findings establish the importance of these secondary oxidative reactions over autoxidation in evaluating the toxicity of HBOCs. We have recently began to monitor the oxidative consequences of administering cell-free hemoglobin (DBBF) into animal model of exchange transfusion. we have paid particular attention to the presence and kietic behaviour of the electron paramagnatic resonance (EPR) detectable species specifically the ferric heme and the protein-based radicals. we found that endogenous ascorbate removes these key percursors to oxidative damage by cell-free hemoglobin both invitro and invivo. Removal of these species would have significant effects on the overall toxicity associated with cell-free hemoglobin. Kinetics of Nitric Oxide Binding to Hemoproteins. Direct cytotoxic effects associated with cell-free hemoglobins (Hb) developed as oxygen therapeutics have been ascribed to redox reactions between Hb and NO. We have investigated the impact of diaspirin cross-linked Hb (DBBF-Hb), a blood substitute, on cell signaling pathways events that are modulated by NO and other biological peroxides (i.e. H2O2, LOOH, and ONOO-) in vitro. Bovine aortic endothelial cells (BAECs) subjected to severe hypoxia expressed hypoxia-inducible factor (HIF-1a) in a time course that paralleled the expressions of phosphotyrosine (pY) and heme oxygenase (HO-1). Co-incubation of the oxy form (HbFe2+) with hypoxic BAECs resulted in an increase in the expression of HIF-1a in a manner that corresponded linearly with the loss of HbFe2+ and accumulation of the ferric form (HbFe3+). Inclusion of HbFe3+ with hypoxic BAECs produced twice as much expression in the HIF-1a and HO-1 proteins as apposed to HbFe2+ alone, and HbFe2+ plus hypoxia. In addition, higher and more persistent levels of the ferryl form (HbFe4+) (due to the consumption of endogenous peroxides) were found in the hypoxic media containing Hb. Nitric oxide (NO) released from an NO donor reduced the levels of HIF-1a in the hypoxic cells treated with either HbFe2+ or HbFe3+, but had little or no effect on the levels of HO-1 and pY proteins. We have concluded that DBBF-Hb modulates key cell-signaling pathways as it converts oxygen signal to a transduction signal that activates HIF-1a, which may result in alterations of important physiological mediators. In human trials with current generation blood substitutes, gastrointestinal discomfort has been universally reported. The symptoms include nausea, vomiting, diarrhea, dysphagia or generalized abdominal pain. These symptoms are believed to be related to NO scavenging by Hb, causing localized spasm throughout gastrointestinal tract. We have recently shown in collaboration with Dr. Baldwin, University of Arizona that bolus injection of some chemically modified hemoglobins can increase venular permeability and mast cell degranulation in the rat mesentery. It was speculated that these hemoglobins extarvasate from the intestinal mucosal capillaries and that DBBF-Hb, in particular causes ultastructural changes in the intestinal epithelium. These changes were found to be correlated strongly with the redox chemistry of each hemoglobin. Interestingly cyanomet DBBF-Hb, in which the iron center is unable to bind NO, and other oxidants, significantly reduced leak numbers and mast cell degranulation. Redox Reactivity of Hemoglobins: search for a safer blood substitute. Oxidative processes, which are in many cases enhanced when modifications are introduced that lower the oxygen affinity, can limit the safety of these HBOC proteins. We have carried out a systematic evaluation of two modified human Hbs (O-R-PolyHbAo and DBBF-Hb) and one bovine Hb (PolyHbBv). We have measured both the oxidative products present in the Hb preparations and followed the oxidative reactions during 37oC incubations. Autoxidation, the primary oxidative reaction which initiates the oxidative cascade is highly correlated with P50 (R = 0.987; p< 0.002). However, when comparing the results for the other oxidative processes, two different classes of oxidative reactions are identified. The formation of oxyferrylHb like the rate of autoxidation increases for all modified Hbs. However, the subsequent reactions, which lead to heme damage and eventually heme degradation are enhanced for the modified human Hbs, but actually suppressed for bovine modified Hbs. The rhombic heme (distorted geometry) measured by electron paramagnetic resonance, which is the initial step that causes irreversible damage to the heme, is found to be a reliable measure of the stability of ferrylHb and the tendency to produce degradation products. DBBF-Hb, a Hb-based oxygen carrier (HBOC), for which toxic side effects have been well documented, has the highest level of rhombic heme (41-fold greater than for HbAo), even though its rate of autoxidation is relatively low. These findings establish the importance of these secondary oxidative reactions over autoxidation in evaluating the toxicity of HBOCs. An extension of these studies; we developed a more reliable rapid mixing method to detect heme degradation products in several chemically modified Hb solutions. We recently described a stopped-flow fluorescence method for the detection of fluorescent heme degradation products in Hb solutions challenged with high concentration of H2O2 and reported the rate constants for these processes. We have also recently reported that DBBF-Hb, and analogue of Baxter's DCLHb, under mild oxidative stress can cause endothelial cells to die apoptotically and neurotically. In this study we clearly documented a role for ferryl-DBBF-Hb as the causative agent of these changes. This project incorporates FY2002 projects 1Z01BQ004022-10 and 1Z01BQ004023-10.